Human papillomaviruses (HPVs) are etiological agents for many human diseases, including genital warts, skin warts, cervical cancer, and certain types of head-and-neck and skin cancers. Although two prophylactic vaccines against HPV infection are presently available and recommended for immunization of young girls/boys, therapeutic vaccines that are most needed for the majority of HPV-infected populations are still lacking. Since early protein 2 (E2) encoded by HPVs is crucial for viral DNA replication, HPV genome segregation, control of viral gene expression, and induction of cellular apoptosis and senescence, it has become a promising target for the development of anti-viral therapeutics. In the last funding period, we have identified bromodomain- containing protein 4 (Brd4) as a central cellular factor mediating diverse functions of HPV-encoded E2 protein, particularly via formation of an E2-Brd4 silencing complex able to block activator protein-1 (AP-1)-triggered gene activation from latent HPV chromatin that exhibits no transcriptional activity. In this renewal application, we propose three aims to dissect the mechanisms of HPV chromatin-dependent transcriptional regulation: 1. To define the mechanism of AP-1-dependent HPV chromatin transcription 2. To define the repression mechanism of E2-Brd4 in HPV chromatin transcription 3. To define HPV-specific versus global cellular gene transcription regulated by AP-1, Brd4, and E2 We will first apply an innovative HPV chromatin transcription system that we have developed to define the role of cellular chromatin-modifying enzymes, including p300 and CBP histone acetyltransferases (HATs), in AP-1-dependent activation of HPV chromatin transcription. We will then dissect the steps of E2-Brd4 silencing complex in suppressing HPV chromatin transcription and further elucidate the functional role of two newly identified Brd4-interacting proteins, i.e., G9a histone methyltransferase (HMT) and topoisomerase II-binding protein 1 (TopBP1), in E2-regulated HPV transcription. To distinguish HPV-specific and global host cell gene transcription, we will also apply genome-wide approaches, in collaboration with Dr. Bing Ren at UC-San Diego, to analyze the transcriptional profiles and binding patterns of these HPV transcriptional regulators and chromatin modifiers, and with the help from Dr. Thomas Kodadek at Florida Scripps, we will utilize synthetic chemistry to develop small compounds blocking Brd4 action in HPV transcription. Our central hypothesis here is that conformational changes in Brd4 provide a molecular switch for Brd4 recruitment of distinct cellular factors to regulate HPV-specific and cellular gene transcription. The combination of mechanistic studies and genome-wide analyses will significantly advance our understanding of viral and cellular factors modulating HPV transcription and facilitate the development of therapeutic agents blocking HPV-induced human diseases.